Biochemistry March 2, 1971
Volume 10, Number 5
@ Copyright 1971 b y the American Chemical Society
Purification and Properties of Pyruvate Phosphate Dikinase from Propionic Acid Bacteria" Herbert J. Evans? and Harland G. Wood$
ABSTRACT : Pyruvate phosphate dikinase has been purified 300-fold from Propionibacterium shermanii grown with glycerol and 85-fold from the organism grown with lactate as the substrates. The enzyme is induced by a factor of 6-10 by growth with lactate as compared to glycerol. The dikinase has optimal activity in the pH range of 6.5-7.0, and requires Mg*+ and a monovalent cation (NHd+ or K+) for maximal activity. The apparent K,,, values observed for the formation of M; P-enolpyruvate from pyruvate are: pyruvate, 1.0 X ATP, 4.3 X M ; Pi, 1.0 X M ; free Mg2+, 4.0 X
P
yruvate phosphate dikinase catalyzes the reaction shown subsequently in eq 1. The enzyme has been observed in propionic acid bacteria by Evans and Wood (1968a,b), in the leaves of tropical grasses by Hatch and Slack (1968), and in the parasitic amoeba, Entamoeba histolytica, by Reeves (1968). The enzyme has subsequently been reported to occur in another plant, Amaranthirs palmeri (Slack, 1968), and another bacterium, Bacteroides symbiosus (Reeves et al., 1968). Hatch and Slack proposed that the enzyme functions in tropical grasses to convert pyruvate to P-enolpyruvate, and that COZ is fixed by combination with the P-enolpyruvate. However, the opposite conversion, P-enolypyruvate to pyruvate, appears to occur in E . histolytica and B. symbiosus, since these organisms lack pyruvate kinase (Reeves, 1968; Reeves et al., 1968). The mechanism of the reaction has been investigated by
* From the Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106. Receiued June 26, 1970. This work was supported by a research grant from the U. S. Atomic Energy Commission (AT-11-1-1783) and a training grant from the National Institutes of Health (2T01-GM-00035). t The data are from the thesis submitted in partial fulfillment of the requirements for the Ph.D. degree from Case Western Reserve University. Present address: Department of Biochemistry, Duke University Medical Center, Durham, N. C. 1 Author to whom to address correspondence.
M; and for the formation 10-8 M; and NH4+, 2.0 X of pyruvate from P-enolpyruvate: P-enolpyruvate, 3.6 X M ; PPi, 1.2 X M ; Mgz+,2.4 X M; AMP, 1.5 X M ; and NH4+,2.1 X M. Under the conditions tested the reaction was very specific for the adenine nucleotides in both directions. The guanine nucleotides inhibit 8 % in the forward reaction and 50% in the reverse reaction. Co2+ and Mn2f will substitute for Mg2+but with reduced velocities. Ca2+, Fe2+, and CuZ+ inhibit the reaction. The s 2 0 1 w value of the enzyme is -6.9 S. The enzyme is cold labile but is stabilized in 1 M sucrose.
Evans and Wood (1968b) and it has been proposed to involve the following sequence. enzyme
Pr + ATP I _ enzyme-PP + AMP1
Pr
enzyme-PP
P
(la)
+ pi J_ enzyme-PP + rPPi
__
(1b)
P + pyruvate enzyme + P-enolpyruvate sum: pyruvate + ATP + Pi J_ P-enolpyruvate + AMP + PPi enzyme-P
(IC)
(1)
Evidence for the scheme includes isolation of a azP-labeled protein after incubation of the enzyme with [ azP]P-enolpyruvate. [14C]Pyruvate exchange into P-enolpyruvate occurs by reaction I C without addition of nucleotides or Mg2+, and [14C]AMP exchange occurs with ATP by reaction l a without an observed requirement for pyruvate or PPi. For the exchange of [azP]Pi into PPi there was a requirement for either ATP or P-enolpyruvate to provide the enzymePPi or enzyme-P for reaction lb. [azP]P-enolpyruvatelabeled the ,&phosphate and [azP]PPi the y-phosphate of ATP as
1
Abbreviations used are definined in Biochemistry 5, 1445 (1966).
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is required for this sequence. Thus far, however, the enzymePPi has not been detected. There may not be a pyrophosphate intermediate but rather two phosphates bonded to the enzyme at two different sites and one of them may be quite labile. Andrews and Hatch (1969) investigated the mechanism of the enzyme from the leaves of sugar cane, and indicated that the reaction proceeds in two steps rather than three. enzyme
+ ATP + Pi I _ enzyme-P + AMP + PPi
(Id)
+ pyruvate I_enzyme + P-enolpyruvate
(IC)
enzyme-P sum: pyruvate
+ ATP + Pi If P-enolpyruvate
+ AMP + PPi
(1)
As evidence against the previously given mechanism (reactions la-c) and for reaction Id, Andrews and Hatch did not observe a P-enolpyruvate-dependent exchange of [ 2P]Pi into PPi, but found an apparent requirement for AMP as well as ATP for the exchange. In addition, they found that the [I4C]AMP exchange into ATP required both Pi and PPi. The results of the exchange studies reported previously (Evans and Wood, 1968b) are consistent with this model, with the exception that a P-enolpyruvate-dependent exchange of [a2P]Piinto PPi was observed with the enzyme from P. shermanii. This exception is difficult to explain by the latter mechanism. The exchanges observed with the enzyme from P . shermanii were slow, but would probably be stimulated by the addition of "a+, since the exchange studies were performed before the requirement for NH4+ was demonstrated. It is possible that the mechanism of the enzyme from plants is different from that of the enzyme from bacteria. The present report deals with the purification and properties of the enzyme from Propionibacterium shermanii. Materials AMP, ATP, trisodium P-enolpyruvate, and 0-DPNH were purchased from Sigma Chemical Co. ; sodium pyruvate and dithioerythritol from Calbiochem; imidazole from Eastman Organic Chemicals; monobasic and dibasic potassium phosphate, KCl, MgC12, NHICl, and sodium pyrophosphate from Baker Chemical Co.; lactic acid from Clinton Corn Processing Co. ; yeast hydrolysate from Yeast Products, Inc. ; (NH&S04, enzyme grade, from Mann Research Laboratories; bovine serum albumin (fraction V) from Armour Pharmaceutical Co. ; and ADP from Pabst Laboratories. Malate dehydrogenase was from Boehringer Mannheim Corp., or was purified from propionic acid bacteria (Allen et a[., 1964). Lactate dehydrogenase, pyruvate kinase, hexokinase, myokinase, and glucose 6-phosphate dehydrogenase were from Boehringer Mannheini. Inorganic pyrophosphatase was a product of Worthington Biochemical Corp. P-enolpyruvate carboxytransphosphorylase was purified from propionic acid bacteria (Lochmiiller et al., 1966; Wood et a/., 1969a). DEAE-cellulose (type 40), TEAE-cellulose, and cellulose phosphate were obtained from Carl Schleicher & Schuell Co. Before use all the celluloses were washed successively with 0.5 N NaOH, distilled water, 0.1 N HCl, distilled water, and 0.05 M potassium phosphate buffer (pH 6.8). Fine particles were removed by centrifugation at 1000 rpm for 5 rnin in an International PR-2 centrifuge. The cellulose was thoroughly equilibrated with the starting buffer by five times repeated suspension in the buffer followed by filtration, until the pH of
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the wash was the same as the starting buffer. Sephadex G-50 and DEAE-Sephadex A-50 were obtained from Pharmacia Fine Chemicals, Inc. Before use they were swelled, washed, and equilibrated as recommended in the product brochures. Methods Pyruvate phosphate dikinase may be assayed by measuring P-enolpyruvate formation from pyruvate or pyruvate formation from P-enolpyruvate. Following purification this was done by linkage with other enzymes but in the initial stages of the purification the dikinase activity was measured by a nonlinked assay. The incubation mixture contained, in micromoles per milliliter: potassium phosphate buffer (pH 7.9), 20; MgC12, 12; ATP, 4; sodium pyruvate, 4; EDTA, 0.1; 2-mercaptoethanol, 2; and in units per milliliter, inorganic pyrophosphatase, 8 ; and the dikinase. The final volume was 0.5 ml. After 10-min incubation at 25", the reaction was terminated by adding 0.3 ml of 10% trichloroacetic acid and after centrifugation, the supernatant solution was neutralized with Tris base. The P-enolpyruvate then was determined in a part of the solution by conversion into malate by the action of Penolpyruvate carboxytransphosphorylase (reaction 3 of Discussion) and malate dehydrogenase, and the oxidation of DPNH was monitored at 340 mp in a Beckman D U spectrophotometer equipped with a Gilford recorder. This assay contained in micromoles per milliliter: potassium phosphate buffer (pH 6.8), 2.7; MgC12, 9.4; KHCO,, 30; P-DPNH, 0.13; CoC12,0.1 ; and in units per milliliter, P-enolpyruvate carboxytransphosphorylase, 0.1, and malate dehydrogenase, 0.5. The linked assay in the direction of formation of P-enolpyruvate involved coupling directly with P-enolpyruvate carboxytransphosphorylase (reactions 1 and 3 of Discussion) and malate dehydrogenase, and determining the rate of DPNH oxidation at 340 mp. The temperature was 25" and the mixture contained, in micromoles per milliliter: potassium phosphate buffer (pH 6.8), 10; MgC12, 22; sodium pyruvate, 2 ; ATP, 2; KHCO,, 30; NHICl, 25; COC12,O.l; P-DPNH, 0.17; and in units per milliliter, P-enolpyruvate carboxytransphosphorylase, 0.3 ; malate dehydrogenase, 15, and the dikinase. A mixture containing all components except enzymes, ATP, and MgC12 was freshly prepared each day and gassed with 100% COz to pH 6.7. All assays were performed with two cuvets, one of which contained all components of the reaction, and another in which ATP was omitted. The latter was necessary because of the presence of contaminating enzymes which oxidized DPNH. I n the direction of pyruvate formation the enzyme was coupled with lactate dehydrogenase. The following concentrations in micromoles per milliliter were optimal : imidazoleC1 buffer (pH 6.7), 50; MgC12,20; NH4Cl,25; P-enolpyruvate, 2; AMP, 2; PPi, 2 ; P-DPNH, 0.17; and in units per milliliter, lactate dihydrogenase, 1.0, and the dikinase. A mixture containing all the components except the enzymes and PPi is stable for 2 days if kept on ice. The assay was performed at 25" with two cuvets, one cuvet containing the complete reaction mixture and the other all but PPi. The control value was subtracted from that observed when PPi was included. Protein concentration was determined by the ultraviolet absorption method (Warburg and Christian, 1941) in the steps of the purification subsequent to the cellulose phosphate column. Prior to this purification the protein was determined by the microbiuret method (Zamenhof, 1957), using bovine serum albumin as a reference standard. All specific activities
FORMATION OF PHOSPHOENOLPYRUVATE
are expressed in the tables as micromoles of P-enolpyruvate formed per minute per milligram of protein. For determination of the stoichiometry of the reaction Penolpyruvate and ADP were determined by the use of pyruvate kinase and lactate dehydrogenase. AMP was determined in the same cuvet as ADP by the addition of myokinase. ATP was determined with hexokinase and glucose 6-phosphate dehydrogenase. P, was determined by the method of Gomori (1942) scaled down by a factor of ten. The Pi samples were read at 660 mp against a blank containing no Pi. PP, was determined in the same way as P, after incubation with 0.5 unit of pyrophosphatase and 5 mM MgC12 for 20 min at 25 '. Growth of Bacteria. P. shermanii (strain 19W) was grown on a glycerol- bicarbonate-phosphate medium (Lochmiiller et al., 1966), or on a lactate medium which contained, per liter of medium, 65 ml of 60% sodium lactate, 2.7 g of KHtPOa, 13.35 g of K2HP04, 3.5 g of yeast hydrolysate, and 1.0 ml of vitamin solution. The vitamin solution contained, per 100 ml of solution, 75 mg of calcium pantothenate, 75 mg of thiamine-HC1, 50 mg of D-biotin, and 1.0 g of Co(NO&. 6H20. The bacteria were adapted to growth on lactate by three or four transfers in this medium after an original inoculation from the glycerol medium. Fermentation was carried out in 15 1. of medium in 20-1. carboys at 30", with daily shaking to avoid clumping of the cells. Large scale fermentations were in a 550-1. vessel at the New England Enzyme Center, Boston, Mass. The cells were harvested by centrifugation in Sharples centrifuges 19 days after inoculation when grown in carboys and 7 days after inoculation when grown in the large fermenter. The yield of bacteria was approximately 3.8 g/l. of medium. Results Purification of Pyruvate Phosphate Dikinase from P. shermanii Grown on Glycerol Medium. The steps used to purify pyruvate phosphate dikinase from P. shermanii grown on glycerol are summarized in Table 1. The first three steps of the purification are identical with those used in the initial steps of purification of oxalacetate transcarboxylase (Wood et a[., 1969b). These steps include breakage of the cells in an Eppenbach colloid mill, DEAE-cellulose adsorption and batch elution, and then elution from a cellulose phosphate column. The protein eluted with 0.05 M potassium phosphate (pH 6.8) was precipitated with ammonium sulfate and used as the source of the dikinase. AMMONIUM SULFATE FRACTIONATION. The ammonium sulfate concentration of the protein solution was estimated from the conductivity as measured with a Barnstead purity meter and then solid ammonium sulfate was added to bring the solution to 35 saturation. After stirring for 1 hr the precipitate was removed by centrifugation at 20,OOOg for 30 min at 0". The supernatant solution then was brought to 55 % saturation, stirred, and centrifuged as before. The same procedure was used to prepare 55-75 fraction in which the majority of the enzyme occurred. ELUTIONFROM DEAE-CELLULOSE. The precipitate from the 55 to 75 fraction was dissolved in phosphate buffer and dialyzed at 4" for 3 hr against three changes of 1 1. of 0.20 M potassium phosphate buffer (pH 6.8) before placing it on a column of DEAE-cellulose equilibrated with the same buffer. The bed volume of the column was approximately 50 cm3/g of protein. The column was washed with the equilibrating buffer until the OD280 fell below 0.1. The protein was then eluted with 200 ml of a linear gradient from 0.2 M potassium
Purification of Pyruvate Phosphate Dikinase from P . shermanii Grown with Glycerol as the Substrate. TABLE I:
Total Sp Units Act.c (pmoles/ (unit/ Method of Fractionation min) mg) Crude extract Batch elution from DEAEcellulose (0.3 M eluate) 35 Elution from cellulose phosphate (0.05 M eluate) Ammonium sulfate satura- 23.0 tion (55-75 %) Elution from DEAE-cel9.0 lulose Extraction with ammo5.2 nium sulfate (60-55 %)
Purifi- Yield cation (%)
0.002 0.003
1 1.5
0.006
3
1000
0.01
5
66
0.20
100
26b
0.60
300
15*
The enzyme at this stage was obtained as a by-product of the purification of transcarboxylase (Wood et a[., 1969b). b One-half of the material from the ammonium sulfate step was used for the elution from DEAE-cellulose and extraction with ammonium sulfate. The values have been corrected to give the amount assuming all the material had been processed. c Activity was determined by the nonlinked assay described in the Methods section.
phosphate (pH 6.8) to 0.2 M potassium phosphate plus 0.2 M KC1 (pH 6.8). The pyruvate phosphate dikinase appeared near the end of the gradient, and it was precipitated with ammonium sulfate at 90% saturation and collected by centrifugation. EXTRACTION WITH AMMONIUM SULFATE: 70, 65, 60, 5 5 , and 50% saturated ammonium sulfate solutions were prepared by diluting saturated ammonium sulfate to the appropriate concentration in 0.10 M potassium phosphate buffer (pH 6.8) containing 0.20 M KC1. The precipitate was successively extracted with each of the above solutions, using 1 ml of ammonium sulfate solution/lO mg of protein in the precipitate. The extractions were performed in a Nalgene centrifuge tube containing a magnetic stirring bar and the suspension was stirred for approximately 15 min and then centrifuged for 15 min at 20,OOOg. After the supernatant solution had been removed, the precipitate was resuspended in the next extraction solution, and the procedure was repeated. The pyruvate phosphate dikinase was associated primarily with the 5 5 % extract which was then precipitated with ammonium sulfate at 75 % saturation and sedimented at 20,OOOg. Purification of Pyruoate Phosphate Dikinase from P. shermanii Grown on Lactate Medium. The amount of the enzyme in crude extracts from cells grown on lactate was found to be 6 to 10 times higher than that in extracts of glycerol-grown cells. The purification of this enzyme is summarized in Table 11. The first four steps were essentially the same as those for cells grown in glycerol medium with the following modifications: 0.40 M potassium phosphate buffer (pH 6.8) was used in place of the 0.30 M phosphate for the final batch elution from DEAE-cellulose and the enzyme activity was found in the fraction from 35 to 55 saturated (NH&S04. ELUTIONFROM TEAE-CELLULOSE. The remaining steps in BIOCHEMISTRY,
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TABLE 11: Purification of Pyruvate Phosphate Dikinase from P. shermanii Grown with Lactate as the Substrate.
Total Units. Sp Act. &moles/ (Units/ Purifi- Yield* Method of Fractionation min) (mg cation (X) Crude extract Batch elution from DEAEcellulose (0.4 M eluate) Elution from cellulose phosphate (0.05 M eluate) Ammonium sulfate saturation (35-5573 Elution from TEAEcellulose Elution from DEAESephadex
1435 2820
0.02 0.05
1 2.5
3680
0.12
6
100
2790
0.20
10
76
900
0.60.
30
24d
520
1.70
85
144
a Purification was from 2.0 kg (wet weight) of P . shermanii grown on lactate. The assay of pyruvate formation from Penolpyruvate was found to be the most rapid and accurate method for locating the enzyme in thc last two steps. The specific activity in the direction of P-enolpyruvate synthesis was then determined on the pooled enzyme. b Yield is based on the activity found after elution from cellulose phosphate, since the greatest number of units are found at that stage. c Thirty per cent of the activity was recovered from the TEAE-cellulose column, but the activity spontaneously increased during storage of the precipitate in 1 M sucrose. The highest specific activity observed after storage was 1.65. d Approximately onethird of the material from the ammonium sulfate step was used for the TEAE-cellulose and DEAE-Sephadex steps. The value has been converted to that which would be obtained assuming all the material was processed.
the purification were performed at room temperature. The precipitate from the 35 to 55z fraction was dissolved in a minimum volume of distilled water and dialyzed against 0.05 M potassium phosphate buffer (pH 6.8) until the conductivity as determined using a Barnstead meter was below that of 0.15 M (NH4)2S04. The solution was then placed on a column of TEAE-cellulose equilibrated with 0.10 M potassium phosphate buffer (pH 6.8), with a bed volume of approximately 60 cma/g of protein. The protein was eluted with 0.20 M potassium phosphate buffer (pH 6.8) until the protein concentration was approximately 0.01 mg/ml. Elution was then begun with 0.20 M potassium phosphate plus 0.20 M KC1 (pH 6.Q and the fractions containing the dikinase were collected, assayed, pooled, and brought to 90% saturation with solid ammonium sulfate. ELUTIONFROM DEAE-SEPHADEX. The precipitated dikinase was dissolved in a minimum volume of distilled water and placed on a column of Sephadex G-50 (fine) equilibrated with 0.10 M potassium phosphate (pH 6.8) using 4-cma bed volumes of Sephadex/ml of protein solution (approximately 80 mgiml). The column was washed with the same buffer and the desalted protein was collected, pooled, and immediately placed on a DEAE-Sephadex A-50 column with a bed volume of approximately 200 cma/g of protein. The protein solution
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Stoichiometry of the Pyruvate Phosphate Dikinase Reacti0n.a
TABLE 111:
Zero Time &moles)
Compound Pyruvate P-enolpyruvate ATP AMP Pi PPi
0 1.84 0 1.79 0.16 3.14
7 min Net Change &moles) (pmoles) 1.12 0.59 1 17 0.64 1.63 2.24
t1.12 -1.25 +1.17 -1.15 +1.47 -0.90
a The incubation contained the following (in micromoles per milliliter): Tris-HC1 buffer (pH 7.4), 50; MgC12, 5; Penolpyruvate, 1.23; AMP, 1.19; PPi, 2.10; P-DPNH, 1.0; and in units, lactate dehydrogenase, 0.8; and dikinase, 0.3 (in 1 % bovine serum albumin). The final volume was 1.5 ml and the reaction was terminated after 7 min with trichloroacetic acid. Pyruvate was determined from the net change in optical density and the other components as described in the Methods section. N o ADP was found before or after the incubation.
was eluted using a convex gradient from 0.15 M potassium phosphate (pH 6.8) to 0.2 M potassium phosphate plus 0.3 M KC1 (pH 6.8), obtained with 500 ml of the former and twice the volume of the latter flowing into it. The fractions containing dikinase were pooled and precipitated with ammonium sulfate and sedimented at 20,OOOg. The protein was dissolved in 1.O M sucrose and stored at - 15". When stored in this way, over a period of months the enzyme tended to spontaneously recover activity which had been lost during the fractionation on TEAE-cellulose. Stoichiometry of the Pyruvate Phosphate Dikinase Reaction. The stoichiometry of the dikinase reaction is shown in Table 111. It is evident that the amounts of P-enolpyruvate, AMP, and PPI used were approximately equimolar with the amounts of pyruvate, ATP, and Pi formed during the reaction. The discrepancy in the values obtained for Pi and PPi may reflect the hydrolysis of some PPi during the determination of Pi. Properties of Pyruvate Phosphate Dikinase. COLDLABILITY. One of the major problems encountered in the purification of the dikinase was the lability of the enzyme. Although the enzyme is relatively stable during the early stages of the purification, it does deteriorate when stored as the ammonium sulfate precipitate at 4" or colder. After the purification on DEAEor TEAE-cellulose the enzyme becomes quite labile when cold. An example of the lability is presented in Table 1V. The dikinase in 0.05 M potassium phosphate (pH 6.8) lost all enzymatic activity over a period of 17 hr at 0". But an identical sample lost only 31 of its activity over the same period at 30". Pyruvate, at 0.7.mM concentration, afforded some protection against cold deterioration. The activity lost by exposure to cold could not be regained by incubation of the enzyme at 30" for prolonged periods. For storage the ammonium sulfate precipitates of the enzyme were dissolved in 1.0 M sucrose and stored at -15O. Enzyme stored in this way retained full activity for at least 6 months. Samples for study of the enzyme were routinely prepared from the enzyme stored in sucrose by dilution in a solution of 1 % bovine serum albumin containing 100 mM imidazole-Cl and 5 mM MgClz at
z
FORMATION OF PHOSPHOENOLPYRUVATE
Effect of Temperature on the Stability of Pyruvate Phosphate Dikinasca
TABLE IV:
Original Activity Remaining (Z) Temp of Storage ("C)
30 0 0 (7 X 10-4
M
pyruvate)
1 hr
4.5 hr
17hr
94 85 87
83 50 74
69
0 25
*Pyruvate phosphate dikinase from the extraction with ammonium sulfate (Table I) was passed through a Sephadex G-50 column equilibrated with 0.05 M potassium phosphate (pH 6.8), assayed by the nonlinked assay and divided into three samples for storage as described. After storage for the indicated time, the samples were warmed for 1 min at 30' and assayed. The stored samples contained 1.8 mg of protein/ml.
pH 6.7. Once prepared, the dilutions were kept at room temperature, and lost activity very gradually and could be used over a period of 2 days. SEDIMENTATION PATTERN. The sedimentation behavior of pyruvate phosphate dikinase which was purified through the DEAE-Sephadex step (Table 11) is shown in Figure 1. Enzyme which has been equilibrated with 0.05 M potassium phosphate buffer (pH 6.8) by passage through a Sephadex G-50 column gave two peaks with sedimentation coefficients of 6.7 and 4.6 S. The enzyme lost 86% of its activity during the preparation and centrifugation. Evidence that the major, faster sedimenting peak contained the enzyme was obtained by partial separation of the proteins of each peak using a partition cell (Yphantis and Waugh, 1956). When the enzyme was equilibrated with 100 m~ imidazoleCI, 50 mM ammonium sulfate, 5 m~ pyruvate, 5 m M MgCll at pH 6.7, a major peak with a sedimentation coefficient of 6.9 S and a small amount of faster moving material were observed (Figure 1B). The faster moving material may indicate the presence of a contaminant or some aggregation of the major component. Assay of the enzyme before and after the centrifugation showed that 9 4 Z of the enzyme activity was retained. ELECTROPHORESIS. Electrophoresis of the enzyme from the last step of Table I1 was performed on polyacrylamide gel at pH 7.4 by a method suggested by Williams and Reisfeld (1964), with 100 p g of the sucrose-stored enzyme. The electrophoresis was at room temperature for 2 hr, followed by staining with Amido Schwartz black for 1 hr. Excess dye was removed by electrophoresis overnight. One major band was observed but there was also a diffuse area of stained material which trailed behind the major band, possibly due to dissociation and reassociation occurring during the electrophoresis. Four minor bands were also visible and these all together were visually estimated to he no more than 5% of the total protein. EFFECT OF pH ON THE REACTION. The effect of p H on the formation of P-enolpyruvate is shown in Figure 2 and on the formation of pyruvate in Figure 3. The optimal pH for the reaction is approximately pH 6.5-7.0 in either direction. NUCLEOTIDE SPECIFICITY.The nucleotide specificity for the formation of P-enolpyruvate and of pyruvate is shown in Table V. Under the conditions of the assay for formation of P-enolpyruvate the requirement for ATP was specific, and
FIGURE 1: Sedimentation patterns of pyruvate phosphate dikinase. The preparation was from the last step of Table 11. A total of 1.38 mg of protein/ml was used for part A which was prepared by passage through a Sephadex G-50 column to remove sucrose and to transfer the enzyme to 0.1 M potassium phosphate (PH6.8). Sedimentation was in the direction of the arrows at 50,740 rpm and 19.2" in a 30-mm double-sector cell. The Schlieren photographs were taken at 16 and 32 min at phassplate angles of M ) and 50", respectively. The observed values are 6.7 and 4.6 S. At the end of the run the enzyme had 14% of the original activity. A total of 2.9 mg of protein/ml was used for part B which was prepared by passage through a Sephadex (3-50 column equilibrated with 0.1 M imidazoleCI (pH 6.7), 0.05 M ammonium sulfate, 0.005 M pyruvate, and 0.005 M MgCh. Sedimentation was in the direction of the arrows at 59,780 rpm and 21.4" in a 12-mm double-sector cell with the equilibration buffer in the other sector. The schlieren photographs were taken at 16 and 32 min at phase-plate angles of 55 and 40", respectively. The observed value of the major peak is 6.9 S. At the end of the run the enzyme had 94% of its original activity.
v: Nucleotide Specificity of Pyruvate Phosphate Dikinase.0
TABLE
Formation of P-enolpyruvate Formation of Pyruvate from from Pyruvate P-enolpyruvate NucleoInhibn tide Act. (%) (%) Nucleotide Act. ATP GTP CTP UTP TTP
100 0 0 0 0
8 0
0 0
AMP GMP CMP UMP TMP
(Z)
Inhibn (Z)
100
0 0 0 0
50
6 0 0
a The activity was determined as described for the linked assays, except for substitution of the nucleotides shown in the table. The inhibition was determined using the indicated nucleotide in addition to ATP or AMP at equimolar concentration. The dikinase was from the TEAE-cellulose step of Table 11.
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2: The effect of pH on the formation of P-enolpyruvate from pyruvate by pyruvate phosphate dikinase. The reactions were in 1.0 ml as described in the Methods except that the mix was gassed with C 0 2 to the indicated pH range and the pH was determined immediately before addition to the cuvet. Each cuvet contained 2.6 pg of dikinase from the TEAE-cellulose step of Table I1 (0.95 specific activity by assay of the formation of P-enolpyruvate), After 2 min, the cuvet contents were transferred to a test tube and the pH was redetermined. The range in pH for the two determinations is represented by the horizontal line. Addition of P-enolpyruvate showed that P-enolpyruvate carboxytransphosphorylase and malate dehydrogenase were not limiting. V is expressed as change in optical density at 340 mp per min x lo3. FIGURE
when other nucleotides were added in equimolar concentration with ATP, GTP was found to inhibit approximately 8%, but the other nucleotides gave negligible inhibition. Likewise in the formation of pyruvate, AMP was specific, and when both AMP and a second nucleotide were added in equimolar concentration, GMP inhibited approximately 50 %, CMP inhibited approximately 6 %,and the other nucleotides were without effect.
'
.
PH FIGURE 3: The effect of pH on the formation of pyruvate from Penolpyruvate by pyruvate phosphate dikinase. The reactions were performed as described in the Methods section, except the following buffers were used in the pH range indicated (all at 0.05 ionic strength): acetate buffer, pH 5.Q-5.8; Tris-maleate, pH 5.65-7.9; Tris-CI, pH 7.15-8.75; sodium potassium phosphate, pH 6.3-7.7; and imidazole-Cl, pH 6.1-7.35. Each cuvet contained 0.86 pg of dikinase from the last step of Table I1 (1.7 specific activity by assay of P-enolpyruvate). Addition of pyruvate showed that lactate dehydrogenase was not limiting. V is expressed as change in optical density at 340 mp per min x 103.
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CMg2+1X
4: The effect of the concentration of Mg*' on the formation of P-enolpyruvate from pyruvate by pyruvate phosphate dikinase. The assays were as described in the Methods section, except for variation of the concentration of Mg2+.The dikinase was from the TEAE-cellulose step of Table I1 (0.95 specific activity). V is expressed as the change in optical density at 340 mp per min X lo3. FIGURE
K, VALUES OF SUBSTRATES AND ACTIVATORS. The apparent K, values for the substrates and activators of the dikinase reaction were determined both for the formation of P-enolpyruvate, and for the formation of pyruvate. The ratio of the concentration of substrate to velocity was plotted against the substrate concentration. This treatment of the data is recommended by Wilkinson (1961), and was found to give the most accurate determination of K , and V,,, with small errors in the observed velocity (Dowd and Riggs, 1965). The intercept of the line with the abscissa gives the -K, value and the intercept with the ordinate is the ratio Km/Vma,.The slope is equal to the reciprocal of V,,,. For formation of P-enolpyruvate the assays were as described in Methods, except for variation of the concentration of the substrate or activator. The dikinase was from the TEAEcellulose step of Table I1 (0.95 specific activity). The apparent M and the V,,, 0.94 ,unole/ K, for pyruvate was 1.0 X M, min per mg of protein. For ATP the K , was 4.3 X and the Vmax0.86, and for phosphate the K, was 1.0 X lop3 M and the V,,, 0.97. The observed K, for phosphate could have been that of the P-enolpyruvate carboxytransphosphorylase since it was used as one of the linking enzymes in the assay. Lochmuller et al. (1966) observed a K, for P, of 1.17 X 1OWa M with carboxytransphosphorylase. Evidence that this was not the case was obtained by varying the Concentration of the carboxytransphosphorylase. If the K, was that of the carboxytransphosphorylase and this enzyme became rate limiting rather than the dikinase, then an increase in the amount of carboxytransphosphorylase should increase the observed rate of reaction at the K, value. This increase was not observed when the amount of carboxytransphosphorylase was tripled. The observed K, is about the same as the apparent K,,, of 5 X 10-4 M found for the dikinase from sugar cane leaves by Hatch and Slack (1968). The effect of the concentration of Mgz' on the velocity of the forward reaction is seen in Figure 4. The reaction was very slow below 1.8 mM, an increase in velocity was observed between 1.8 ana 7 mM, and inhibition above 7 mM. The anomalous behavior below 1.8 mM can be explained if the reaction requires free Mgz+, since essentially all the Mg2+ would be
FORMATION OF PHOSPHOENOLPYRUVATE
bound to ATP until the Mg2i concentration exceeded the ATP concentration, because of the high association constant of the Mg-ATP complex (Burton, 1959). The plot of the ratio of the concentration of free Mg2+ to velocity us. the conM, centration of free Mgz+ gave an apparent K, of 4.0 X and a Vma, of 1.3 pmoles/min per mg of protein. The association constant of 2.25 x given by Burton was used to calculate the concentration of free Mgz+. Although carboxytransphosphorylase also required MgZ+,the apparent K, for M (Lochmuller et a/., 1966) which is lower Mg2+ is 1.2 X than that observed for the dikinase. The inhibition of the reaction at concentrations of Mg2+above 7 mM may be due to a competitive inhibition of free Mg2+for the site where MgATP or Mg-phosphate binds to the enzyme. REQUIREMENT OF MONOVALENT IONS. Pyruvate phosphate dikinase from B. symbiosus was reported by Reeves et al. (1968) to require NH4+ ion, and the observed apparent K, was 2.5 x 10-8 M. A similar stimulation has been observed with the dikinase from P. shermanii. The apparent K , was found to be 2.0 X 10-3 M, and the V,,, was 0.78 pmole/min per mg of protein. Although KT would substitute for NH4+, the apparent K, for K+ was not determined. The K , determinations for the formation of pyruvate were performed as described in Methods except for variation of the concentration of the substrate or activator. The dikinase was from the last step of Table I1 (1.7 specific activity). The apparent K , for P-enolpyruvate was 3.6 X M and the V,,, was 2.5 pmoles/min per mg of protein. The apparent K , given by Reeves et a/. (1968) for the dikinase from B. symbiosus is 6 X 10-5 M and Andrews and Hatch (1969) give a value of 1.1 X 10-4 M for the apparent K , of the dikinase from the leaves of sugar cane. The apparent K , for AMP was 1.5 X M and V,,, was 2.0; Reeves et al. (1968) reported a K, of “less than 0.01 m” and Andrews and Hatch (1969) repc-’ed no decrease in the reaction rate at 4 X M. For PPi the K, was 1.2 X M, compared to 1 X M reported by Reeves et a/. (1968) and 4.0 X 10-5 M reported by Andrews and Hatch (1969). The V,,, was 3.3. For Mg2+ the K , was 2.4 X M and the anomalies with Mg*+ seen in Figure 4 for the formation of P-enolpyruvate were not observed. The M and the V,,, was 4.2. The K, for NH4+ was 2.1 X 3.5. Within a factor of three the apparent K,’s of the dikinase from P. shermanii agree with those published for the dikinase from sugar cane (Hatch and Slack, 1968; Andrews and Hatch, 1969), and with those published for the dikinase from B. symbiosus (Reeves et al., 1968). The fact that the values of K, and Vmax are dependent on the concentrations of the other substrates and activators may explain the differences b e t ~ e e nthe values given in this report and those of the other investigators. REQUIREMENT OF DIVALENT METAL. Various divalent metals were substituted for Mg2+ to determine if any could replace the requirement of Mg2+. Only the formation of pyruvate was studied because the P-enolpyruvate carboxytransphosphorylase used in the assay for P-enolpyruvate formation also has a requirement for a divalent metal. The results are shown in Table VI. Of the metals tested, Mn2+and Co2+,and possibly Zn2+,were able to activate the reaction, but not as well as Mg2+. Precipitation occurred when higher concentrations of metals were tested. The apparent K, for activation by Coz+ was 5 X M, which is approximately fivefold lower than the apparent K, of Mg2+, but the V,,, obtained with Coz+ was only 1.65 pmoles/min per mg of protein, which is considerably lower than the value with Mg2+. Ca2+,Cu2+,and Fe2+inhibited the dikinase reaction. Inhibi-
v,,,
Requirement of Divalent Metals by Pyruvate Phosphate Dikinase:
TABLE VI:
Mg2+ Mg2+ Ni2+ c o 2+ Coz+
+ CaZ+
+ Ca2+
Mg2+ Mg2+ Mgz+ MnZ+ Zn2+
+ Cu2+ + Fe2+
100 60 0 48 9
25 25 0.5 8 2.4 2.4 0.5
+
+
5.9 5.9 3.0 5.9 3.0
100 33 89 12 4
+ 0.6 + 0.6
40
82 67 11
a The data in the upper portion of the table were obtained using the conditions as described in the text for assay of pyruvate formation with the indicated cations substituted for MgZ+.The cations other than Mg2+ were present in the highest concentration which could be reached without formation of a precipitate. MnZ+, Zn2+, and Ba2+ all caused precipitates at 0.4 mM and therefore are not included in this section of the table. Each cuvet contained 1.7 pg of dikinase from the last step of Table 11. The data in the lower part of the table were obtained using a modified assay. The mixtures contained in micromoles per milliliter: Tris-C1 (pH 7.4), 45; P-enolpyruvate, 3; PPi, 3; AMP, 3; 0-DPNH, 0.12; the indicated cations; and, in units per milliliter, lactate dehydrogenase, 2.4; and 1.4 pg of dikinase from the TEAE-cellulose step of Table 11.
tion by Ca2+ is characteristic of a type I1 metal function, as described by Cohn (1963). In those cases reviewed by Cohn, the type I1 metal is bound to the enzyme and may form a bridge between the enzyme and substrate molecules. Discussion The propionibacteria obviously catalyze the conversion of pyruvate into P-enolpyruvate since they grow on pyruvate and the mechanism of this conversion was the subject of these studies. When P-enolpyruvate carboxytransphosphorylase was discovered by Siu and Wood (1962), it appeared that Penolpyruvate might be formed by the following reactions in analogy with the synthesis in liver by pyruvate carboxylase and P-enolpyruvate carboxykinase (Keech and Utter, 1963).
+ ADP + Pi oxalacetate + P P ~IfP-enolpyruvate + COP + Pi
pyruvate
+ ATP + COz
sum: pyruvate
oxalacetate
+ ATP + PPi If P-enolpyruvate
+ ADP + 2 Pi
(2) (3)
(4)
In liver, P-enolpyruvate carboxykinase catalyzes a reaction similar to reaction 3 except it uses GTP instead of PPi. Thus ATP plus GTP is required rather than ATP and PPi. In support of reaction 4 synthesis of P-enolpyruvate from pyruvate was observed with ATP and PPi by Wood et a[. (1966) using pyruvate carboxylase from avian liver and carboxytransphosB I O C H E M I S T R YV O , L . 10,
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phorylase from propionibacteria. This led to a search for pyruvate carboxylase (reaction 2) in propionic acid bacteria. Synthesis of oxalacetate from pyruvate, ATP, and COZ was observed with extracts of propionibacteria, but there was a requirement for Pi which was not explained by reaction 2. Further study showed that the synthesis of oxalacetate from pyruvate was not uia reaction 2 but occurred by the following reactions. pyruvate
+ ATP + Pi
P-enolpyruvate sum: pyruvate
+ AMP + PPi oxalacetate + PP,
(3)
+ AMP + 2PPi
(5)
P-enolpyruvate
+ C 0 2 + pi
+ ATP + COz + 2Pi oxalacetate
(1)
Cooper and Kornberg (1965) had observed that the synthesis of P-enolpyruvate from pyruvate occurs in E. coli as shown in reaction 6 and is catalyzed by an enzyme named P-enolpyruvate synthase. pyruvate
+ ATP
P-enolpyruvate
+ AMP + Pi
(6)
It appeared (Wood, 1968) that the same reaction occurred in propionibacteria but it then was found that P, was required for reaction 1 as well as for reaction 3. The synthase apparently involves a hydrolytic cleavage in reaction lb, whereas the dikinase involves a phosphorolytic cleavage, thus yielding PP, instead of Pi. Reeves et al. (1968) determined the equilibrium of the dikinase reaction using enzyme from B. symbiosus and obtained a value of 1140 for Keq at pH 7.0 in the direction of pyruvate synthesis. Unlike B. symbiosus and E. histolytica, which lack pyruvate kinase, propionibacteria contain a large amount of pyruvate kinase (unpublished results) and it seems unlikely that the dikinase functions physiologicallyin propionibacteria to form pyruvate from P-enolpyruvate. If a pyrophosphatase were linked with the dikinase in propionibacteria it would overcome the unfavorable equilibrium for P-enolpyruvate synthesis. A preliminary investigation has shown that this enzyme is present in P. shermanii. The equilibrium of the carboxytransphosphorylase reaction is in favor of oxalacetate formation (Wood et al., 1966). Therefore the formation of oxalacetate cia reaction 5 would be favorable when linked with pyrophosphatase, or when linked with other reactions of metabolism which remove oxalacetate. That pyruvate phosphate dikinase functions physiologically for formation of Penolpyruvate is indicated by the 6- to 10-fold increase of the enzyme when the bacteria are grown with lactate instead of glycerol as a substrate. Growth on lactate requires formation of P-enolpyruvate from pyruvate as opposed to growth on glycerol where P-enolpyruvate is formed uia phosphoglycerate. The purification of the dikinase from P. shermanii is difficult because of the instability of the enzyme. The enzyme is not only cold labile, but also loses activity even if the purification is performed at room temperature. Addition of EDTA, dithiothreitol, pyruvate, and MgClz alone and in various combinations does not prevent the loss of activity during purification. Another source of difficulty is the small quantity of the enzyme present in the extracts of the bacteria, thus requiring the processing of a large amount of bacteria to obtain significant yields. Pyruvate phosphate dikinase from P. shermanii has both similarities to and differences from the dikinases purified from
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Zea mays (Hatch and Slack, 1968) and E. histolytica and B.
symbiosis (Reeves et al., 1968). The cold lability of the dikinase from propionibacteria is similar to that observed with the plant enzyme and this loss of activity is not dependent on freezing. In contrast the enzyme from B. syrnbiosus was reported to lose only 10% of its activity over a 2-week period at 4". Another similarity between the enzyme from propionibacteria and the plant enzyme is the requirement for free Mgz+ in the reaction toward P-enolpyruvate. However the dikinase from propionibacteria has a requirement for a monovalent cation just as does the enzyme from B. symbiosis, but this requirement was not observed with the enzyme from plants or the amoeba. Another major difference from the plant enzyme is the optimum pH. It is 8.3 for plants whereas it is 6.5-7.0 for propionibacteria. It is noted that aspartate transaminase and P-enolpyruvate carboxylase were used in the assay of the enzyme from plants. The transaminase has an optimal pH of 8.0-8.5 and P-enolpyruvate carboxylase from peanut cotyledons also has been shown to have an optimal pH of 8.0-8.2 (Maruyama et a/., 1966). Perhaps these coupling enzymes become limiting at lower pH values. Andrews and Hatch (1969) observed that the velocity of the reaction in the direction of P-enolpyruvate synthesis was about six times the velocity of the reverse reaction at pH 8.3. The enzyme from P. shermanii also shows a higher rate of synthesis of P-enolpyruvate than of pyruvate at pH values between 8 and 8.5, but at its optimum pH (6.5-7.0) the rate in the direction of formation of pyruvate is the most rapid. The K, values of the enzyme from different sources do not differ greatly.
References Allen, S. H. G., Kellermeyer, R. W., Stjernholm, R . L., and Wood, H. G. (1964),J . Bacteriol. 87, 171. Andrews, T. J., and Hatch, M. D. (1969), Biochem. J . 114, 117. Burton, K. (1959), Biochem. J . 71,388. Cohn, M. (1963), Biochemistrj, 2,623. Cooper, R. A., and Kornberg, H. L. (1965), Biochim. Bi0phj.s. Acta 104, 620. Cooper, R. A., and Kornberg, H. L. (1967), Biochim. Biophys. Acta 141,211. Dowd, J. E., and Riggs, D. S. (1965), J . Biol. Chem. 240, 263. Evans, H. J., and Wood, H. G. (1968a), Fed. Proc., Fed. Amer. SOC. Exp. Biol. 27, 588. Evans, H. J., and Wood, H. G. (1968b), Proc. Nut. Acad. Sci. U . S. 61, 1448. Gomori, G. (1942),J . Lab. Clin. Med. 27,955. Hatch, M. D., and Slack, C. R. (1968), Biochem. J . 106, 141. Keech, D. B., and Utter, M. F. (1963), J . Biol. Chem. 238, 2609. LochmGller, H., Wood, H . G., and Davis, J. J. (1966), J. Biol. Chem. 241, 5678. Maruyama, H., Easterday, R. L., Chang, H. C., and Lane, M. D. (1966), J. Biol. Chem. 241,2405. Reeves, R. E. (1968),J. Biol. Chem. 243,3202. Reeves, R. E., Menzies, R. A., and Hsu, D. S. (1968), J. Biol. Chem. 243, 5486. Siu, P. M. L., and Wood, H. G. (1962), J . Biol. Chem. 237, 3044. Slack, C. R. (1968), Biochem. Biophys. Res. Commun. 30, 483. Warburg, O., and Christian, W. (1941), Biochem. 2. 310, 384. Wilkinson,G. N. (1961), Bi0chem.J. 30,824. Williams, D. E.,and Reisfeld, R. A. (1964), Ann. N . Y. Acad. Sci. 121,373.
MALATE DEHYDROGENASE FROM
Chlorella pyrenoidosa
Wood, H. G. (1968), J. Viraminol. 14, 59. Wood, H. G., Davis, J. J., and Lochmiiller, H. (1966), J. Biol. Chem. 241,5692. Wood, H. G., Davis, J. J., and Willard, J. M. (1969a), Methods Enzymol. 13,297.
Wood, H. G., Jacobson, B., Gerwin, B. I., and Northrop, D. B. (1969b), Methods Enzymol. 13,215. Yphantis, D. A., and Waugh, D. F. (1956), J. Phys. Chem. 60,630. Zamenhof, S . (1957), Methods Enzymol. 3,696.
Purification and Properties of Malate Dehydrogenase from Chlorella pyrenoidosa. Catalytic Mechanism of the Particulate Form* Cheryl Thomas Stromeyer,? Francis E. Cole,$ and Pierre C. Arquembourg
ABSTRACT : By means of ammonium sulfate fractionation and column chromatography, particulate and supernatant forms of malate dehydrogenase (MDH) from Chlorella pyrenoidosa have been purified to specific activities of 33 and 84, respectively. The two forms have been shown to be chromatographically, electrophoretically, and immunologically different, each showing a characteristic MDH activity when chromatographed on DEAE-cellulose, when electrophoresed on cellulose-acetate, and when developed by Ouchterlony double diffusion. Both forms have a molecular weight of approximately 61,000 as determined by Sephadex gel filtration. The kinetic properties of the particulate MDH were studied by: (1)
T
he malate dehydrogenase system (L-malate:DPN oxidoreductase, EC 1.1.1.37) of animal, plant, and microbial origin has been shown to generate a complex electrophoretic pattern. Two MDH’ proteins, one associated with the supernatant fraction of the cell and the other with the particulate fraction, can generally be demonstrated in animal and microbial species; several plant systems have been suggested to consist of three MDH proteins (Mukerji and Ting, 1969; Ting, 1968; Yamazaki and Tolbert, 1969). Physical properties such as subunit composition (Wolfenstein et al., 1969), fluorescence spectra (Cassman and Englard, 1966a), amino acid composition (Kitto and Kaplan, 1966), and immunological reactivities (Henderson, 1968) have been examined for a number of highly purified animal MDH enzymes. In a few animal species, kinetic studies have led to the elucidation of catalytic mechanisms: either a basic compulsory ordered addition (“ordered bi-bi”) mechanism or some variation thereof has been suggested in each case. But in plant species, though multiple forms of MDH from several systems (Ting, 1968; Mukerji and Ting, 1969) have been stud-
* From the Departments of Biochemistry and Medicine, Louisiana State University Medical Center, New Orleans, Louisiana 701 12. ReceioedJune 17, 1970. This work was supported in part by NSF Grant GB-8317. t This paper represents partial fulfillment of the requirements for the Master of Science Degree. 3 Correspondence should be sent to Dr. F. E. Cole, Department of Biochemistry, Louisiana State University Medical Center, New Orleans, La. 701 12. Abbreviation used is: MDH, malate dehydrogenase.
analysis of initial velocity patterns in the direction of DPNH oxidation at pH 7.4 in 0.1 M potassium phosphate buffer; (2) determination of product inhibition patterns with DPNf and malic acid as product inhibitors; and (3) analysis of inhibition patterns of monoethyl oxaloacetic acid with respect to DPNH and oxaloacetic acid. All three studies yielded results suppor tive of a compulsory ordered addition mechanism for the M and K , particulate MDH. The K , of DPNH of 4 X of oxaloacetic acid of 5 X lod5M as well as the substrate inhibition by oxaloacetic acid at levels above 8.48 X l W 4 M for the particulate MDH are data similar to those obtained with animal mitochondrial malate dehydrogenases.
-
ied in terms of physical and kinetic properties, including thermal inactivation and molecular weight determinations and electrophoretic, chromatographic, and pH profiles, no catalytic mechanism has been defined. Examining the MDH system of Chlorella pyrenoidosa, Cole and his associates (1968) have demonstrated the existence of multiple electrophoretic forms. This communication reports the partial purification of MDH activities from this algal species and includes molecular weight determinations as well as immunological and electrophoretic studies. A detailed kinetic analysis of that M D H activity associated with the particulate fraction of the cell is presented. Experimental Procedures
Organism and Culture Conditions. Steady-state cultures of the high-temperature strain (7-1 1-05) (Sorokin and Myers, 1953) of C . pyrenoidosa were cultured as described previously (Cole et al., 1968). Purification. Cells were harvested by centrifugation (12,100g ) at 4’. The cell pellet, washed twice with 0.1 M Tris-HC1 buffer, pH 8.6, and twice with distilled water, was recovered in 50 ml of 0.2 M potassium phosphate buffer with 0.3 M sucrose, pH 6.5. The sample was mixed with a one-third volume aliquot of glass powder in a 125-ml beaker and sonicated with the blunt tip probe of a Branson Sonic Power Sonifier, Model M52, operated on high power for a total of 3-5 min, in 15-sec periods. The beaker was immersed in an acetone-ice bath and the sample maintained at 5’. After sonic disruption, the sample was centrifuged at 480g for 5 min to sediment only B I O C H E M I S T R Y , VOL.
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